Method of modulating leukemic cell and eosinphil activity with monoclonal antibodies

ABSTRACT

A method of isolating a monoclonal antibody capable of inhibiting any one of IL-3, GM-CSF and IL-5 binding to the common receptor β c , or a monoclonal antibody capable of inhibiting the cytokines binding to a receptor analogous to β c . The method includes the steps of immunizing an animal with a cytokine receptor or portion of a cytokine containing the critical binding site which portion includes the extracellular domain 4 or analogous domain in the analogous common receptor or part thereof. Antibodies producing cells from the animal are then isolated and fused with a myeloma cell line and then screened for a cell line that produces an antibody of the desired type. A monoclonal antibody, or fragments thereof capable of inhibiting the binding of the cytokines IL-3, GM-CSF and IL-5 to the β c  receptor, and a hybridoma cell line producing the antibody are also claimed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 09/762,963filed Feb. 14, 2001, now U.S. Pat. No. 6,720,155 which is a 35 U.S.C §317 of a PCT/AU99/00659, which claims priority from Australia PP5251,filed Aug. 13, 1998. The entire disclosure and contents of the abovepatents and applications are hereby incoporated by reference.

FIELD OF THE INVENTION

This invention relates to a method of isolating monoclonal antibodyinhibitors and reagents derived therefrom and other inhibitors ofcytokine binding including monoclonal antibodies and reagents derivedtherefrom and small molecules capable of inhibiting binding of GM-CSF,IL-3 and IL-5 to the common beta receptor subunit.

INTRODUCTION

Human interleukin (IL)-5, IL-3 and granulocyte-macrophagecolony-stimulating factor (GM-CSF) are cytokines involved in hemopoiesisand inflammation (Metcalf; 1986). All three cytokines stimulateeosinophil production, function and survival (Metcalf; 1986) andtherefore have the ability to influence inflammatory diseases such asasthma, atopic dermatitis and allergic rhinitis where the eosinophilplays a major effector role. IL-5, being the eosinophil specificcytokine, has received most of the initial attention with IL-5 mRNA andprotein levels noted to be elevated in lung tissue and bronchoalveolarlavage (BAL) fluid from symptomatic asthma patients (Fukuda et al 1994).Correlation between IL-5 levels and allergen challenge and diseaseactivity have also been seen (Sur et al, 1996). It is becoming apparent,however, that not only IL-5 but also GM-CSF and IL-3 play a role ineosinophil production and activation in asthma as there is evidence ofboth GM-CSF and IL-3 being synthesized at sites of allergic inflammation(Bagley et al, 1997b; Allen et al 1997). It is possible that expressionof these cytokines contributes to the total number of infiltratingeosinophils and the degree of eosinophil activation. Alternatively, theymay be responsible for different phases of eosinophil infiltration.Recent kinetic data from patients undergoing antigen challenge showedthat IL-5 levels increased between days 2-7 post challenge, whilstGM-CSF peaked at day 2, and remained elevated throughout day 16.Furthermore, GM-CSF detection extended beyond the site of allergenchallenge.

IL-S, GM-CSF and IL-3 stimulate eosinophils and other normal and cancercells by binding to cell surface receptors that comprise aligand-specific α chain and a β chain which is shared by the threereceptors (β_(c)) (Bagley et al 1997a). Binding to each receptor α chainis the initial step in receptor activation, however, engagement ofeither α chain alone is not sufficient for activation to occur.Recruitment of β_(c) by each ligand: α chain complex follows, a stepthat has two major functional consequences: firstly, it allows thebinding of IL-5, GM-CSF and IL-3 to become essentially irreversible; andsecondly, it leads to full receptor activation (Bagley et al 1997a).Since β_(c) is the major signalling component of these receptors itsengagement leads to the activation of JAK-2, STAT-5 and other signallingmolecules culminating in the full plethora of cellular activitiescommonly associated with either IL-5, GM-CSF and IL-3 stimulation suchas eosinophil adherence, priming for degranulation and cytotoxicity, andprolongation of viability (Bates et al, 1996).

In order to block or antagonize the activity of eosinophil-activatingcytokines in vivo three major approaches are being tried. One of themutilizes antibodies to the implicated cytokines. For example, antibodiesto IL-5 are being used in an animal model of allergen-induced asthma andhave shown to have a relatively long lasting effect in preventingeosinophil influx into the airways and bronchial hyperresponsiveness(Mauser et al, 1995). A second approach relies on IL-5 or GM-CSF mutantswhich can bind to the respective α chains with wild type affinity butwhich have lost or shown reduced ability to interact with human β_(c).IL-5 mutants such as E13Q, E13K and E13R, and the human GM-CSF mutantE21R directly antagonize the functional activation of eosinophils byIL-5 or GM-CSF respectively (Tavernier et al 1995; McKinnon et al 1997;Hercus et al 1994b). However, at least in the case of E13K, eosinophilsurvival is not antagonized and in fact this mutant is able to supporteosinophil survival (McKinnon et al 1997). A third approach involves theuse of soluble receptor α chains which can sequester circulatingcytokines. However, this carries the risk of a cytokine:receptor α chaincomplex potentially interacting with surface-expressed β_(c) andtriggering receptor activation. The common theme amongst theseapproaches is that they tackle a single receptor system involving eitherIL-5, GM-CSF or IL-3 leaving the other two eosinophil-acting cytokinesunaffected. Although the concomitant administration of IL-5 and GM-CSFantagonists may be considered, this may be clinically impracticable.

An alternative approach to blocking eosinophil-activating cytokinesinvolves targeting the common β chain of their receptors. Although β_(c)does not directly bind IL-5, GM-CSF or IL-3 alone, it does bind to thesecytokines complexed to the appropriate receptor α chain. Lopez et al inWO 97/28190, which is incorporated herein by reference in its entirety,have identified the major binding sites of, β_(c) for the IL-5:IL-5Rα,GM-CSF:GM-CSFRα and IL-3:IL-3Rα complexes. Significantly, these sitesare utilized by all three complexes and comprise the predicted B′-C′loop and F′-G′ loop in β_(c). Thus targeting β_(c) is not only desirablebut also feasible, with the added potential to allow the simultaneousinhibition of IL-5, GM-CSF and IL-3 action by a single agent. Theseworkers have shown that certain mutants in the B′-C′ and the F′-G′ loopfail to bind IL-5, GM-CSF and IL-3.

SUMMARY OF THE INVENTION

The present invention results from the isolation of a monoclonalantibody BION-1) raised against the membrane proximal domain (domain 4)of β_(c) which is able to block the production and activation of humaneosinophils stimulated by IL-5, GM-CSF or IL-3 and blocks the growth ofleukaemic cell lines. This MoAb was able to block the high affinitybinding of all three cytokines to eosinophils by binding to residues inthe predicted B′-C′ and F′-G′ loops of β_(c), and prevented receptordimerization and β_(c) phosphorylation. It was found that raising anantibody capable of blocking the binding of all three cytokines waspossible by screening monoclonal antibody-expressing hybridoma celllines arising from immunising mice with cells expressing only domain 4of β_(c) and lacking domains 1 to 3 and expressing domain 4 and thetransmembrane and cytoplasmic regions.

Additionally this finding is likely to have implications for othermembers of the cytokine receptor superfamily some of which are sharedsubunits in a given subfamily (that is they bind several cytokines), andsome which are ligand specific and bind to only one cytokine. Thereceptor α-chains for GM-CSF, IL-3 and IL-5 and β_(c) belong to therapidly expanding cytokine receptor superfamily. Within this superfamilyseveral sub-families are now emerging that are characterized by thesharing of a communal receptor subunit by multiple ligands: gp 130 actsas an affinity converter and signal transducer for IL-6 (Hibi et al.,1990; Taga et al., 1992), IL-11 (Hilton et al., 1994), oncostatin M (Liuet al., 1992), ciliary neurotrophic factor, leukaemia inhibitory factor(LIF) (Ip et al., 1992) and cardiotrophin-1(Pennica et al., 1995); theLIF receptor (LIFR) also binds ciliary neurotrophic factor (Davis etal., 1993), cardiotrophin-1 (Pennica et al., 1995) and oncostatin M inaddition to LIF (Gearing et al., 1994); IL-2R β supports affinityconversion and signalling of IL-2 and IL-15 (Giri et al., 1994); IL-2R γchain affinity converts IL-2 (Takeshita et al., 1992), IL-4 (Russell etal., 1993), IL-7 (Noguchi et al., 1993), IL-9 (Kimura et al., 1995) andIL-15 (Giri et al., 1994); evidence also suggests that IL-4 and IL-13share a receptor component (Zurawski et al., 1993) and this subunit hasrecently been cloned (Hilton et al., 1996). It is not known whichresidues in gp 130, LIFR and IL-2R β and γ chains are important forligand binding or indeed whether different ligands share or have uniquesets of binding determinants on these communal receptor subunits.Because these common subunits are vital for transducing signals byseveral ligands, the possibility arises that interfering with theability of these common subunits to bind ligand or to form homodimersmay affect the action of more than one ligand.

Clear similarities in structure between β_(c) and other cytokinereceptors have been recognised and similarities in at least part of thebinding site, the F′-G′ loop, have been identified in Lopez et al in WO97/28190. Accordingly it is an expectation that the means employed bythe inventors to obtain a monoclonal antibody that inhibits binding ofIL-3, GM-CSF and IL-5 will also lead to the isolation of monoclonalantibodies that inhibit binding of other cytokines to their respectivereceptors.

In a broad form of a first aspect the invention could be said to residein a method of isolating a monoclonal antibody capable of inhibiting anyone of IL-3, GM-CSF and IL-5 binding to the common receptor β_(c), or amonoclonal antibody capable of inhibiting a cytokines binding to areceptor analogous to β_(c), said method comprising the step ofimmunising an animal with a cytokine receptor or portion of a cytokinereceptor containing the critical binding site which portion mightinclude the extracellular domain 4 or analogous domain in the analogouscommon receptor or part thereof, isolating antibody producing cells fromsaid animal and fusing antibody producing cells with a myeloma cellline, screening for a cell line that produces an antibody of the desiredtype.

The immunisation may involve introducing a cDNA clone of a portion of orall of the common receptor including the extracellular domain 4 oranalogous domain in the analogous common receptor or part thereof, intoa cell and proliferating said cells to form a recombinant cell line,inoculating an animal with said recombinant cell line, isolatingantibody producing cells from said animal and fusing the antibodyproducing cell line with a myeloma cell line to form a hybridoma cellline, screening for a hybridoma cell line that produces an antibody thatbinds to the recombinant cell line but not to the parent, and thentesting for inhibition against all three cytokines. In one form the cellinto which the cDNA clone is introduced is mammalian and one commonlyused mammalian cell line is a COS cell.

The cDNA may encode a full or partial portion of domain 4 when it is ina configuration where the F′-G′ loop and/or the B′-C′ loop is in itsnative shape. The data below show that cDNA encoding substantially onlydomain 4 of the extracellular portion of β_(c) as well as thetransmembrane and the intracellular portions maintains these sites in asufficiently integral conformation so that an antibody raisedthereagainst will give the inhibition sought. It is postulated that thesame will be the case for analogous receptors for the cytokinesuperfamily. This method should be distinguished from immunising withthe whole receptor since the extracellular domain 4 is likely to becovered or masked by other domains in the whole receptor.

β_(c) has two repeats of the cytokine receptor module (CRM), each ofwhich has two discrete folding domains (CRDs), so that in total β_(c)has 4 domains hence named domains 1 to 4 (β1 to 4). It is postulatedthat domain 2 of any CRM may be an equivalent of domain 4 and thereforedomain 2 may be used in the immunisation.

In addition the domain 4 of β_(c) or equivalent domain in other cytokinereceptors may be expressed in isolation in a microbial host such asEscherichia coli and used to immunise animals for developing monoclonalantibodies.

The analogous receptor may be any one of the cytokine superfamilyreceptors but not limited to the group comprising β_(c), LIFR, gp130,IL-2Rβ, IL-4R/IL-13R, IL-2Rγ, IL-3Rα, EPOR, TPOR and OBR.

It will be understood that in one specific form of this aspect of theinvention the method is used to isolate a monoclonal antibody thatinhibits cytokine binding to a common receptor subunit. The commonreceptor is envisaged to be selected from the group of receptors actingfor more than one cytokine including but not limited to gp130, LIFR,IL2Rβ/IL2Rα, and EL-4R/IL-13R in addition to β_(c).

It will also be understood that the invention encompasses monoclonalantibodies or fragments thereof produced as a result of this first formof the invention.

In a broad form of a second aspect the invention could be said to residein a monoclonal antibody, or fragments thereof capable of inhibiting thebinding of the three cytokines IL-3, GM-CSF and IL-5 to the β_(c)receptor.

The degree of inhibition may range from complete inhibition to moderateinhibition, which inhibition will of course be dependent on the amountof monoclonal antibody or fragments thereof added to inhibit and therelative affinity of the antibody or fragment thereof to the β_(c).

The extent of inhibition of respective ones of the three cytokines isnot necessarily identical and may vary, so the different cytokines maybe inhibited from binding to different degrees.

The antibody fragments may be larger portions such as Fab fragments ormuch smaller fragments of the variable region. These fragments may beused as separate molecules or alternatively may form part of arecombinant molecule which is then used for therapeutic purposes. Thusfor example the monoclonal antibody may be “humanised” by recombiningnucleic acid encoding the variable region of the monoclonal antibodywith nucleic acid encoding non-variable regions of human origin in anappropriate expression vector.

The inhibition preferably leads to blocking of at least one function ofall three cytokines. One of the benefits that is proposed to be derivedfrom these antibodies or antibody fragments is their use in modifyingcells stimulated by one of the three cytokines, and more in one specificform modifying the activity of the three cytokines is proposed to impactgreatly on eosinophil function. Therefore preferably the activity leadsto inhibition of stimulation of effector cell activation and where theantibody or fragment thereof is to be used for treatment of asthma leadsmost preferably to inhibition of IL-5, IL-3 & GM-CSF mediated eosinophilactivation. It will be understood however that cells other thaneosinophils are also the effectors of adverse conditions in humans andanimals as a result of stimulation by these cytokines and inhibition ofsuch stimulation is also contemplated by this invention. These includecells that express either one or all of GM-CSF, IL-3 and IL-5 receptors,the stimulation of which leads to pathology. Examples of these areleukaemic cells, endothelial cells, breast cancer cells, prostate cancercells, small cell lung carcinoma cells, colon cancer cells, macrophagesin chronic inflammation such as rheumatoid arthritis, dendritic cellsfor immunosuppression and neutrophils in inflammation.

Thus in one form the invention may be said to reside in an inhibitor ofleukaemic cell growth wherein the inhibitor is capable of inhibiting thebinding of one or all of IL-3, GM-CSF and IL-5 to the β_(c) receptor.The inhibitor may be BION-1 or an agent capable of inhibiting BION-1binding with β_(c).

A number of different facets of eosinophil function might be modified sothat in one form IL-5, IL-3 & GM-CSF mediated eosinophil survival isinhibited or blocked. In a second form IL-5, IL-3 and GM-CSF mediatedeosinophil activation is inhibited or blocked.

In one form of this second aspect of the invention the monoclonalantibody or fragment thereof binds to at least the F′-G′ loop of domain4 of the β_(c) subunit.

In an alternative form the monoclonal antibody or fragment thereof bindsto at least the B′-C′ loop of domain 4 of the β_(c) subunit but thisalternative form is not limited to monoclonal antibodies or fragmentsthereof that only bind to the F′-G′ loop but includes monoclonalantibodies or fragments thereof that perhaps binds to both the F′-G′ aswell as the B′-C′ loop of domain 4 of the β_(c).

It is thought that the monoclonal antibody isolated by the inventorsinhibits dimerisation of the common receptor units and thus theinvention might encompass an antibody or fragments thereof of the secondaspect of the invention that inhibit β_(c) receptor dimerisation.

In one very specific form the monoclonal antibody is the antibodyproduced by the hybridoma cell line BION-1 (ATCC HB-12525).

In a broad form of a third aspect the invention could be said to residein a hybridoma cell line capable of producing a monoclonal antibody ofany form of the first or second aspect of the invention.

In one specific form of the third aspect of the invention the hybridomacell line is BION-1 (ATCC HB-12525).

Since GM-CSF, IL-3 and IL-5 need to bind their respective a chainsbefore being able to interact with β_(c), at present most screening fornew inhibitors utilise cell-based assays where both, α and β_(c)receptor units are co-expressed. Solid phase assays rely on inhibitionof GM-CSF, IL-3 or IL-5 to their respective a chain only since thesecytokines cannot bind to β_(c) alone. Since BION-1, unlike these threecytokines, can directly bind to β_(c) we propose that it can be used asa novel solid phase screening assay. Any compound that binds theappropriate site which is likely to inhibit all three cytokines willalso inhibit the binding of BION-1. Additionally once further inhibitorycompounds are uncovered these could be used in the place of BION-1 inthat screening process. This therefore facilitates the screening oflarger number of candidate inhibitor compounds.

In a broad form of a fourth aspect therefore the invention could be saidto reside in a method of screening peptides, oligonucleotides and othersmall molecules for their capacity to competitively inhibit the bindingof BION-1 or the binding of an agent capable of inhibiting BION-1binding, to the β_(c) subunit.

Generally the screening assay involves contacting BION-1 or fragmentthereof with the β_(c) subunit or fragment thereof as well as acandidate inhibitory compound, and measuring the degree of binding.

A reporting means is preferably provided to facilitate the detection ofbinding of BION1 or fragment thereof with β_(c) subunit or fragmentthereof. Thus, for example, a competitive binding assay using labelledBION-1 could be used for this purpose. β_(c) or domain 4 of β_(c) isimmobilized on a plate or tube and several compounds added, followed bylabelled or tagged BION-1 or fragments thereof. Since BION-1 binds theregion of β_(c) involved in binding all three cytokines, any compoundsthat block or reduce the binding of BION-1 or fragments thereof to β_(c)or domain 4 will be considered candidate inhibitory compounds. Thus, theavailability of BION-1 as an agent that for the first time allows thedirect binding to the cytokine binding region of β_(c) affords a noveltest for the identification of simultaneous inhibitors of GM-CSF, IL-3and IL-5. It will be understood that the same will apply for othercytokines and their respective receptors.

It will be understood that not the entire β_(c) subunit needs be used toscreen candidate compounds, and certainly the present data indicatesthat a fragment of the β_(c) subunit encompassing domain 4 hassufficient structure in common with the native β_(c) subunit to reflectthe configuration of the cellular target for an inhibitor useful for anin vivo effect.

In a broad form of a fifth aspect, the invention could be said to residein a cytokine inhibitor capable of simultaneously blocking the bindingof β_(c) by IL-3, GM-CSF, and IL-5 made according to the fourth aspectof the invention.

It is thought that compounds that inhibit binding of the IL-3, IL-5 andGM-CSF to the β_(c) will be therapeutically useful for intervention inconditions where IL-3, GM-CSF and IL-5 play a pathogenic role, mainlyallergy, asthma, acute and chronic myeloid leukaemias, lymphoma andinflammation including rheumatoid arthritis, breast cancer and prostatecancer.

Similarly for other common cytokine receptors it is thought thatantagonists or agonists will be therapeutically useful. gp130 isfunctionally analogous to β_(c) in that it is a common binding sub-unitand signal transducer for the IL-6, oncostatin M (OSM), ciliaryneutrotrophic factor (CNTF), leukaemia inhibitory factor (LIF) andIL-11. It is suggested that raising an antibody against a domainanalogous to domain 4 of β_(c) will also lead to blocking of two or moreof these cytokines. Antagonism of this receptor system will be useful ininflammation, leukaemia and lymphoma. Antagonists to IL2Rβ/IL2Rα may beuseful as immunosuppresants. Antagonists of LIFR may be useful for theprevention of implantation of embryos in uteri. Antagonists of IL4/L-13will inhibit IgE production and may be useful in treating asthma andallergies.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Flow cytometry analysis of the staining of MoAb BION-1(continuous line) and an isotype matched IgG₁ control MoAb (dotted line)to (A) COS cells transiently transfected with β_(c), (B) CHO cellsconstitutively expressing β_(c), (C) TF-1.8 cells, (D) neutrophils, (E)eosinophils and (F) monocytes.

FIG. 2. MoAb BION-1 recognizes β_(c) protein. (A) Immunoprecipitation ofβ_(c) from ¹²⁵I-surface labelled CHO β_(c) cell lysate. Both experimentswere performed on 7.5% SDS PAGE under reducing conditions. (C) MoAbBION-1, but not another anti-β_(c) MoAb 1C1, recognises a β_(c) mutant(β_(c)-ΔQP) that contains only domain 4 in the extracellular regions byimmunoprecipitation of CHO ΔQP cells. This mutant has a flag attached soit can also be seen by anti-flag MoAb M2. The experiment was performedon 10% SDS PAGE under reducing conditions.

FIG. 3. Dose-dependent competition for the binding of ¹²⁵I-IL-5 (50 pM),¹²⁵I-GM-CSF (50 pM) and ¹²⁵I-IL-3 (200 pM) by MoAbs BION-1 (•), ananti-β_(c) MoAb control ( ), and an IgG₁ control MoAb (o) to TF-1.8cells (2×10⁶ per point). ( - - - ) represents ligand binding in thepresence of 200-fold excess unlabelled ligand. Each point is the mean oftriplicate determinations.

FIG. 4. A Fab fragment of BION-1 blocks high affinity binding of IL-5,GM-CSF and IL-3. Binding of ¹²⁵I-IL-5 (50 pM), ¹²⁵I-GM-CSF (50 pM) and¹²⁵I-IL-3 (200 pM) were assessed on TFI.8 cells (2×10⁶ per point) in thepresence or absence of 3000 nM MoAb BION-1, or control anti-β_(c) MoAb,or 4200 nM Fab fragment of BION-1 for 2 h at room temperature. 1C1 wasused as a non blocking anti-β_(c) control. Cells were separated fromunbound radioligand by spinning through FCS and the resulting cellpellet was counted. The results are expressed as a percentage of thetotal specific cpm bound seen in the absence of antibody. Non-specificbinding was determined in the presence of 200 fold excess of coldligand. Total binding seen for ¹²⁵I-IL-5, ¹²⁵I-GM-CSF and ¹²⁵I-IL-3 were9744.2, 2567.1 and 3379.13 cpm respectively. Blocking with Fab fragmentof BION-1 was determined from a single point and BION-1 and 1C1 valuesare the mean of duplicate determinations with error bars representing 1standard deviation.

FIG. 5. BION-1 blocks high affinity binding of IL-5, GM-CSF and IL-3 tohuman eosinophils. Human eosinophils (1.8×10⁶ per point) were incubatedwith ¹²⁵I-IL-5 (10 pM or 3 pM), ¹²⁵I-GM-CSF (50 pM) or ¹²⁵I-IL-3 (200pM) either alone or in the presence of 1 μM MoAb at room temperature for2 h. MoAb's 9E10 and 8E4 were used as isotype matched and non-blockinganti-β_(c) control respectively for BION-1. Cells were separated fromunbound radioligand by spinning through FCS and the resulting cellpellet was counted. The results are expressed as a percentage of thetotal specific cpm bound seen in the absence of antibody. Non-specificbinding was determined in the presence of 200 fold excess of cold jigandand was determined to be an average of 0.3% of total counts added. Totalbinding seen for ¹²⁵I-IL-5, ¹²⁵I-GM-CSF and ¹²⁵I-IL-3 were 765, 622 and748 cpm respectively. Each point is the mean of duplicate determinationsand error bars represent 1 standard deviation.

FIG. 6. MoAb BION-1 recognizes an epitope in β_(c) comprising at leastresidues M363, R364, E366 and R418. Human β_(c) wild type and mutants(Woodcock et al, 1996) were tested for reactivity with MoAb BION-1 and1C1 used as a control. Following expression of wild type β_(c) and β_(c)mutants on COS cells, β_(c) was immunoprecipitated by MoAb 8E4, followedby Western blotting by MoAb BION-1 (top) or the non blocking MoAb 1C1(bottom).

FIG. 7. The binding of ¹²⁵I-labelled MoAb BION-1 (1 nM) to TF. 1 cellsis inhibited by IL-3 (•), but not by TNF-α(∘). ( - - - ) Representsinhibition in the presence of 200-fold excess of unlabelled BION-1.

FIG. 8. BION-1 IgG selectively inhibits IL-5, GM-CSF and IL-3 mediatedproliferation of TF 1.8 cells. The proliferation experiments representthe comparison of inhibition of BION-1 IgG at maximal dosage (400 μg/mlBION-1 with IL-5 and IL-3 and 850 μg/ml with GM-CSF) against ED⁵⁰concentrations for IL-5 (0.3 ng/ml), GM-CSF (0.03 ng/ml) or IL-3 (0.3ng/ml). An anti-β_(c) antibody and an irrelevant IgG antibody were usedas controls. The results are expressed as DPM. Each value represents themean of triplicate determinations and error bars represent the SEM.

FIG. 9. Fab fragment of BION-1 and BION-1 IgG inhibits IL-5, GM-CSF andIL-3 mediated proliferation of TF1.8 cells. Intact IgG or Fab fragmentof BION-1 were titrated against a fixed concentration of IL-5 (0.3ng/ml), GM-CSF (0.03 ng/ml) or IL-3 (0.3 ng/ml) in proliferation assayswhere TF1.8 cells at 5×10⁴/well were incubated for 48 hours and thenpulsed for 5 hours with 0.5 μCi/well ³H-Thymidine. The results areexpressed as DPM. Each value represents the mean of triplicatedeterminations and error bars represent the SEM.

FIG. 10. Eosinophil survival. (A) Viability of eosinophil after 36 hoursin the presence of IL-5, IL-3 and GM-CSF. (B) Viability of eosinophilafter 36 hours in the presence of IL-5, IL-3 and GM-CSF (1 nM) anddifferent concentrations of MoAb BION-1 (∘) and 8E4 (•). Each point isthe mean of triplicate determinations from three samples and error barsrepresent 1 standard deviation.

FIG. 11. MoAb BION-1 inhibits IL-5-stimulated CD69 up-regulation onhuman eosinophils. (A) CD69 up-regulation in the presence of differentconcentrations of IL-5, IL-3, GM-CSF and TNF-α. (B) CD69 up-regulationstimulated by 1 nM of IL-5, GM-CSF, IL-3 or TNF-α in the presence ofdifferent concentrations of MoAb BION-1 or control anti-β_(c) MoAb 8E4.Each point is the mean value of three replicates and error barsrepresent 1 standard deviation.

FIG. 12. Inhibition of L-3-induced α and β chain dimerization andphosphorylation by MoAb BION-1. Immunoprecipitations using anti-IL-3RαMoAb 9F5 or anti-β_(c) MoAb 8E4 from M07e cells preincubated with MoAbsBION-1, MoAb 1C1 or medium alone (−) for 1 min, before being stimulated(+) or not (−) with IL-3 (50 nM) for 5 min. The figure was visualised byPhosphorImaging and the position and molecular weight (in thousands) ofmarker proteins are shown to the left of the gels. The gels werereprobed by Western blotting analysis using anti-phosphotyrosine MoAb3-365-10 and the top panel shows the image of part of the gels in theβ_(c) area.

FIG. 13. Screening peptides for inhibition of MoAb BION-1 binding tosoluble β_(c) domain 4 adsorbed to solid phase. E. coli derived solubleβ_(c) domain 4 (sβ_(c)#4) was coupled to Maxisorp ELISA plates at 10μg/1 ml in 0.1M carbonate buffer overnight and then blocked with 1% BSA.(A) B45.pep (FHWWWQP-GGGCDYDDDK) (+) and (B) YB12.pep(FPFWYHAHSPWS-GGGCDYKDDDK) (•) were derived from biopanning librarieswith sβ_(c)#4 using an acid eluant. B45 was allowed the bond to sβ_(c)#4at 0.0125 μM and YB12 was allowed to bind sβ_(c)#4 at 0.025 μM. BION-1was added to the plates at a starting concentration of 5 μg/ml andserial dilutions were used to titrate the BION-1 down to 0.004 μg/ml.The plate was washed again and BION-1 binding to sβ_(c)#4 was detected.

FIG. 14. BION-1 specifically inhibits the growth in vitro of chronicmyelomonoctic cells (CMML). A contol antibody (ICI) does not inhibit.

DETAILED DESCRIPTION OF THE INVENTION

Materials and Methods

ΔQP cDNA: To express domain 4 of β_(c) on the cell surface we cloned theactivated β_(c) mutant, hβ_(c)ΔQP, with an extracellular deletionremoving domains 1 to 3 (D'Andrea et al 1996), into the eukaryoticexpression vector pcDNA3 (Invitrogen).

Cytokines and cell lines: Recombinant human IL-3 and GM-CSF wereproduced in E. coli as described (Barry et al 1994, Hercus et al 1994b).Recombinant human IL-5 was purified from E. coli by Bresatec (Adelaide,South Australia). Recombinant EPO was purchased from Johnson & Johnson(New Jersey). TNFα was a gift from Dr. J. Gamble in the Hanson Centrefor Cancer Research. COS cells were transfected with receptor cDNA asdescribed previously (Woodcock et al 1994). CHOβ_(c) and CHOΔQP cellsstably expressing either full length β_(c) or domain 4 respectively weregenerated by electroporation (Hercus et al, 1994a). TF1.8 cells were agift from Dr J. Tavernier from University of Gent, Belgium. MO7e cells,a human megakaryoblastic cell line, were from Dr P Crozier, Aukland, NewZealand. Human eosinophils were purified from the peripheral blood ofslightly eosinophilic volunteers via sedimentation through dextran andcentrifugation through a discontinuous density gradient of hypertonicMetrizamide, as previously described (Vadas et al 1979). Eosinophilswere more than 92% pure. Human neutrophils and monocytes were purifiedfrom peripheral blood as described previously (Lopezet al, 1990) withmore than 95% purity.

Generation of anti-β_(c) MoAbs: BALB/c mice were immunizedintraperitonally with 1×10⁷ COS cells transfected with β_(c) or ΔQPexpression constructs. ΔQP constructs express substantially only domain4 of the extracellular domains of β_(c). The immunizations were repeated4 times at two-weekly intervals. Four weeks after the finalimmunization, a mouse was boosted with 2×10⁶ COS transfectantsintravenously. Three days later, splenocytes were harvested and fusedwith NS-1 myeloma cells as previously described (Sun et al 1996).Hybridoma supernatants were screened on CHO β_(c) or CHO ΔQP cells byflow cytometry, with untransfected CHO cells as a control. Allantibodies were from single hybridoma clones as selected by limitingdilution method. MoAbs were purified from ascites fluid or hybridomasupernatant by a protein A sepharose column. The isotypes of MoAbs weretested with a Mouse MoAb Isotyping Kit (Boehringer Mannheim, Germany).Fab fragments were generated using a Fab Preparation kit (Pierce,Rockford, Ill.) following the supplied protocol.

Immunofluorescence: Freshly purified neutrophils, eosinophils,monocytes, or CHO and COS cell transfectants (5×10⁵) were incubated with50 μl of hybridoma supernatant or 0.25 mg of purified MoAb for 45-60 minat 4° C. Cells were washed twice and then incubated with FITC-conjugatedrabbit anti-mouse Ig (Silenus, Hawthorn, Victoria, Australia) foranother 30-45 min. Cells were then washed and fixed before analysingtheir fluorescence intensity on an EPICS-Profile II Flow Cytometer(Courter Electronics). Two colour staining was carried out by additionalincubation with another MoAb directly coupled to PK.

Ligand Binding Assay: IL-3 and GM-CSF were radio-iodinated by the iodinemonochloride method (Contreras 1983). ¹²⁵I-IL-5 was purchased fromDupont NEN (North Sydney, NSW, Australia). Binding assays were performedas previously described (Lopez et al 1989). Briefly, 1-2×10⁶ TF-1.8cells were preincubated with BION-1 Fab fragments, anti-β_(c) or controlMoAbs over a concentration range of 0.06 to 4200 nM for 1 hour.Radio-labelled ligand was then added and incubated for a further twohours before the cells were separated from free label by spinningthrough FCS. Counts associated with the resulting cell pellets weredetermined by counting on a γ counter (Cobra Auto Gamma; PackardInstruments Co, Meridien, Conn.). Non-specific binding was determinedfor each ligand by binding in the presence of a 200 fold excess ofunlabelled cytokine.

MoAb binding assay: MoAbs were radio-iodinated by the chloramine-Tmethod (McConahey 1980). Saturation binding studies were performed byincubating 2×10⁶ TF1 cells in a range of concentrations ofradio-labelled antibodies in the presence or absence of excessunlabelled antibodies. The binding affinity of each anti-β_(c) MoAb toits antigen was determined by Scatchard transformation (Scatchard 1949)and analysed with the ligand program (Munson and Rodbard, 1980).Competition binding experiments were set up by preincubating the TF1.8cells with a range concentration of IL-3, or GM-CSF, or IL-5 prior toadding radio-labelled MoAb QP1 for two hours as per ligand bindingassay. Epitope analysis was determined by testing the capacity of eachunlabelled MoAb to compete for the binding of each radio-labelled MoAbto the β_(c) on COS cell transfectant.

Co-immunoprecipitation of α and β chains and the β_(c) phosphorylationassays: M07e cells were surface labelled with ¹²⁵I by thelactoperoxidase method as described previously (Walsh and Crumpton,1977). The labelled cells incubated in either medium containing IL-3(100 ng/ml) alone or IL-3 together with the MoAb QP1, 1C1 (0.5 mg/ml) or7G3 (30 mg/ml) for 5 min. Cells were lysed in lysis buffer consisting of137 mM NaCl, 10 mM Tris-HCl (pH 7.4), 10% Glycerol, 1% Nonindet P40 withprotease and phosphatase inhibitors (10 mg/ml leupeptin, 2 mMphenylmethlysulphonyl fluoride, 10 mg/ml aprotonin and 2 mM sodiumvanadate) for 30 min at 4° C. followed by centrifugation of the lysateat 10,000×g for 15 min to remove cellular debris. The lysate wasprecleared with mouse-Ig-coupled Sepharose beads for 18 h at 4° C. andincubated with anti-IL-3Ra, anti-β_(c) MoAb beads for 2 hr at 4° C. Thebeads were washed 6 times with lysis buffer and immunoprecipitatedproteins were separated by SDS-PAGE under reducing condition. Theimmunoprecipitated proteins were detected by a Phosphorlmager (MolecularDynamics, Sunnyvale, Calif.). The gels were then reprobed by Westernblotting analysis with an anti-phosphotyrosine MoAb, 3-365-10(Boehringer Mannheim, Frankfurt, Germany).

TF-1.8 cell proliferation assay: TF-1.8 cells were grown in the presenceof 2 ng/ml of GM-CSF. The cells were starved for 24 hours before settingup proliferation assays as described previously (Sun et al., 1996). Fromdose-response curves the half-maximal proliferation dosage of IL-3 (0.3ng/ml), GM-CSF (0.03 ng/ml), IL-5 (0.3 ng/ml) or EPO (5 ng/ml) waschosen to perform proliferation experiments in the presence of a rangeof concentrations of MoAbs. The ³H-Thymnidine incorporation of eachsample was determined by liquid scintillation and expressed asdisintegrations per minute (DPM).

Eosinophil survival assays: The maximal dose of IL-5 required to supporteosinophil survival after 36 hours was determined. Eosinophils were thencultured with 1 nM of IL 5 plus anti-β_(c) MoAbs for 36 hours. Theviability of eosinophils was quantitated by propidium iodide stainingand flow cytometry analysis as described (Nicoletti, 1991).

CD69 expression: CD69 expression on eosinophils was-measured by means ofan antiCD69 monoclonal MoAb coupled to PE by flow cytometry.

β_(c) mutants and MoAb Mapping: Single amino acid substitutions in theB′-C′ and F′-G′ loops of domain 4 of the β_(c) have been describedpreviously (Woodcock, et al., 1994; 1996). The cDNAs for wild type β_(c)and each of the β_(c) mutants in the B′-C′ and F′-G′ loops wereintroduced into COS cells by the electroporation (Hercus et al., 1994).Cell transfectants were analysed for surface expression with 48 hoursafter transfection. Mutants on the B′ and C′ β-strands such as L356N,W358N, 1374N and Y376N were expressed on FDCP1 cells from retroviralexpression constructs (Jenkins et al., 1995). Epitope-mapping of anti-scantibodies was analysed by Immunofluorescent study. The anti-β_(c) MoAbswere tested for their abilities to recognise wild type β_(c) and theβ_(c) mutants analysed by flow cytometer using standardimmunofluorescence method. For each mutant, the experiment was repeatedat least twice.

BION-1 binding inhibitory peptides: E. coli derived soluble β_(c) domain4 (sβ_(c)#4) was coupled to Maxisorp ELISA plates at 10 μg/1 ml in 0.1Mcarbonate buffer overnight and then blocked with 1% BSA. B45.pep(FHWWWQP-GGGCDYDDDK) was derived from four rounds of biopanning thePh.D-7mer library with sβ_(c)#4 using an acid eluant. YB12.pep(FPFWYHAHSPWS-GGGCDYKDDDK) was derived from biopanning the Ph.D-12merlibrary with sβ_(c)#4 using an acid eluant. B45 was allowed the bond tosβ_(c)#4 at 0.0125 μM and YB12 was allowed to bind sβ_(c)#4 at 0.025 μM.The plate was washed in TBS+0.5% Tween. BION-1 was added to the platesat a starting concentration of 5 μg/ml and serial dilutions were used totitrate the BION-1 down to 0.004 μg/ml.

The plate was washed again and BION-1 binding to sβ_(c)#4 was detectedwith α-mouse conjugated to HRP, using a colour based reaction which wasread on a plate counter by absorption.

BION-1 inhibition of chronic myelomonocytic cells: Peripheral blood froma patient with chronic myelomonocytic leukemia was centrifuged overFicoll-Paque to separate the mononuclear cells. After washing andcounting, the cells were plated on agar as a concentration of 10⁵ perplate. After incubation in medium containing monoclonal antibodiesBION-1 or 1C1, with or without IL-3, for 14 days at 37° C. the number ofarising colonies were counted by mycroscopical examination. Each cellcluster containing more than 40 cells was counted as a colony.

Results

Development of MoAb BION-1

Previous experiments have shown that the putative F′-G′ loop of β_(c)contains a common binding site for IL-5, GM-CSF and IL-3 (Woodcock etal, 1996; WO 97/28190). We have now produced a blocking compound,represented by MoAb BION-1, by immunizing mice with COS cellstransfected with a cDNA coding for domain 4 of β_(c). Screening ofhybridoma supernatants was performed on a CHO cell line expressingdomain 4 of β_(c). One hybridoma cell line was identified which produceda MoAb which specifically recognized this cell line and not a parentalCHO cell line not expressing domain 4 of β_(c). This MoAb was termedBION-1 and was characterized in biochemical, binding and biologicalexperiments.

BION-1 Recognizes Domain 4 as Well as Wild Type β_(c).

MoAb BION-1 was tested for reactivity against cell lines transfectedwith β_(c) and against primary cells known to express IL-5, GM-CSF andIL-3 receptors. BION-1 recognized COS cells transiently transfected withβ_(c) CHO cells permanently transfected with β_(c), the erythroleukaemicTF-1 cell line, and purified peripheral blood human neutrophils,eosinophils and monocytes (FIG. 1).

The antigen recognized by BION-1 was confirmed to be domain 4 of β_(c),by biochemical analysis of transfected cells. FIG. 2A shows that BION-1immunoprecipitated a surface ¹²⁵I-labelled protein of about 120,000 MWconsistent with the size of β_(c). Similarly, BION-1 recognized aprotein of 120,000 MW by Western blotting using lysates of CHO cellsexpressing full length wild type β_(c) (FIG. 2B). The size of thesebands also corresponded to the bands recognized by a previouslydeveloped anti-β_(c) MoAb (Korpelainen et al 1993; Woodcock et al,1996). To formally show that BION-1 recognized domain 4 of β_(c) we alsotested BION-1 for its ability to immiinoprecipitate domain 4 expressedon the surface of CHO cells. As a positive control we incorporated ashort polypeptide to the N-terminus of domain 4 (flag epitope) to whicha MoAb has been previously developed. As a negative control, we used theanti-β_(c) MoAb 1C1 which recognizes an epitope located elsewhere inβ_(c). FIG. 2C shows that BION-1 immunoprecipitated a band of about80,000 MW from ¹²⁵I-surface labelled-domain 4-expressing CHO cellsconsistent with the expected size of domain 4. MoAb M2 against the flagepitope added to domain 4 of β_(c) also precipitated a similar sizeprotein. In contrast, MoAb 1C1 failed to immunoprecipitate domain 4.These experiments show that BION-1 can specifically recognize domain 4of β_(c) on the surface of cells and following denaturation of theprotein.

BION-1 Inhibits the High Affinity Binding of IL-5. GM-CSF and IL-3 toTF-1 Cells and to Human Eosinophils

Given that domain 4 of β_(c) is crucial for the high affinity binding ofIL-5, GM-CSF and IL-3, we examined whether BION-1 was able to affectthis binding. We found that BION-1 inhibited in a dose-dependent mannerthe binding of ¹²⁵I-IL-5, ¹²⁵I-GM-CSF and ¹²⁵I-IL-3 to the humanerythroleukaemic cell line TF-1. For each radioligand we used thesmallest possible concentration to maximize the possibility of measuringhigh affinity. This can be more readily achieved with IL-3 and GM-CSFfor which the difference between the low affinity component (provided byeach α chain alone) and the high affinity component (provided byco-expressing β_(c) with each a chain) is about 1,000 fold and 30 foldrespectively. In the case of IL-5, the affinity conversion of β_(c) isonly in the 25 fold range, hence, high and low affinity binding cannotbe clearly separated. This is likely to explain why BION-1 showscomplete inhibition of ¹²⁵I-GM-CSF and ¹²⁵I-IL-3 binding (FIG. 3). Theresidual ¹²⁵I-IL-5 binding seen with high concentrations of BION-1 islikely to be the result of low affinity ¹²⁵I-IL-5, binding (α chain)which BION-1 would not be expected to inhibit. This is consistent withBION-1 inhibition of ¹²⁵I-IL-5, binding reaching a plateau beyond whichno further inhibition can be detected (FIG. 3 a). Other anti-β_(c) MoAb(anti-,β_(c) control) and the IgG₁ MoAb control did not inhibit¹²⁵I-IL-5, ¹²⁵I-GM-CSF and ¹²⁵I-IL-3 binding to TF-1 cells (FIG. 3).

The blocking effect of BION-1 was seen whether the MoAb was used aspurified IgG or as Fab′ fragment. FIG. 4 shows that the Fab′ fragment ofBION-1 blocked the binding of 50 pM ¹²⁵I-IL-5, 50 pM ¹²⁵I-GM-CSF and 200pM ¹²⁵I-IL-3 to TF-1 cells.

Since one of the major clinical utilities of blocking IL-5, GM-CSF andIL-3 binding is likely to be in asthma, a disease in which eosinophilsare believed to play a major role, it was important to test whetherBION-1 could block the binding of IL-5, GM-CSF and IL-3 to these cells.As shown in FIG. 5, BION-1 inhibited the binding of all threeradio-labelled cytokines to purified human eosinophils. In contrast,other anti-β_(c) MoAb or the IgG₁ MoAb control failed to do so.

Epitope Mapping of BION-1

The fact that BION-1 inhibited the binding of ¹²⁵I-IL-5, ¹²⁵I-GM-CSF and¹²⁵I-IL-3 to TF-1 cells and eosinophils suggested that it might bebinding to the critical region in β_(c) to which these cytokines bind orat least in close proximity to it. To try to define the region/epitopein β_(c) recognized by BION-1, we used several mutants of β_(c) andexamined whether substitutions of individual amino acids in thepredicted B′-C′ loop or F′-G′ loop impaired BION-1 binding. Two sets ofexperiments were carried out. In the first instance weimmunoprecipitated wild type β_(c) from transfected COS cells with aMoAb anti-β_(c). The immunoprecipitates were then tested for reactivitywith the control anti-β_(c) MoAb 1C1, or BION-1. The results shows thatβ_(c) mutants carrying the substitutions M363A/R364A, or E366A or R418Awere not recognized by BION-1 (FIG. 6). In a second set of experiments,the direct binding of radio-labelled BION-1 was measured ontransfectants expressing the same mutants. Similar results were obtainedin that whilst 1C1 bound with similar affinity to wild type β_(c) andthe β_(c) mutants, BION-1 binding was eliminated by the M363A/R364A,E366A and R418A mutants (Table I). These results suggest that theepitope recognized by BION-1 is formed, at least in part, by M363 and/orR364, E366 and R418. This is consistent with the disclosure in WO97/28190 that agents that bind the putative F′-G′ loop (of which R418 ispart of) will be antagonists of IL-5, GM-CSF and IL-3.

BION-1 and IL-3 Reciprocally Inhibit Each Other's Binding

To confirm that the epitope recognized by BION-1 was the same or closeto the binding site utilized by IL-5, GM-CSF and IL-3, we performed thereverse experiment, in which BION-1 was radio-labelled and increasingconcentrations of IL-3 used to compete for ¹²⁵I-BION-1 binding. Theresults showed (FIG. 7) that IL-3 competed for ¹²⁵I-BION-1 binding in adose-dependent manner emphasizing the close and intimate proximity ofBION-1 and IL-3 binding epitopes in β_(c)

BION-1 Specifically Inhibits the Function of IL-5. GM-CSF and DL-3Including Their Stimulation of Eosinophil Production and Activation.

To ascertain whether the inhibition of IL-5, GM-CSF and IL-3 binding byBION-1 was translated into inhibition of IL-5, GM-CSF and IL-3stimulation we used the factor dependent TF-1 cell line. This cell lineproliferates in the presence of either IL-5, GM-CSF, IL-3 orerythropoietin (EPO) (FIG. 8). As shown in FIG. 8 MoAb BION-1 but notother MoAb anti-β_(c) nor an IgG, control MoAb inhibited the stimulationof TF-1 cell proliferation by IL-5, GM-CSF and IL-3. In contrast, thestimulating ability of erythropoietin was not inhibited showingspecificity of BION-1 for the IL-5/GM-CSF/IL-3 receptors system.

Titration experiments showed that BION-1 inhibited cytokine-mediatedTF-1 cell proliferation in a dose-dependent manner with an ED₅₀ of about100-300 nM (FIG. 9). FIG. 9 also shows that other anti-β_(c) MoAb werenot inhibitory, and that Fab fragments of BION-1 behaved similarly toBION-1 as a whole IgG with virtually overlapping ED₅₀ values.

Since eosinophils are believed to be the major effector cells in asthmaand they respond to IL-5, GM-CSF and IL-3, we examined BION-1 for itsability to block eosinophil production, eosinophil survival andeosinophil activation in response to these three cytokines. We foundthat BION-1 but not MoAb 8E4 inhibited the ability of IL-5, GM-CSF andIL-3 to stimulate the formation of eosinophil colonies from human bonemarrow cells (Table II).

Importantly, BION-1 inhibited the pro survival activity of IL-5, IL-3and GM-CSF on purified peripheral blood human eosinophils. Whilst thesecytokines are essential for maintaining eosinophil viability (FIG. 10A),blocking of β_(c) by MoAb BION-1 promotes eosinophil cell death tolevels similar to those observed in the absence of cytokines (FIG. 10B).Eosinophils can be activated by IL-5, GM-CSF and IL-3 as well as bytumour necrosis factor (TNF-α), a factor that operates through the TNF-αreceptor. A sign of eosinophil activation is the upregulation of theCD69 surface antigen, a phenomenon induced by all four cytokines (FIG.11A). Using this activation system we found that BION-1 inhibited theactivation of eosinophils by IL-5, GM-CSF and IL-3 (FIG. 11B). OtherMoAb anti-β_(c) or IgG₁controls failed to do so. In addition theblocking effect of BION-1 was found to be specific in that thestimulating activity of TNF-α was not inhibited (FIG. 11B).

BION-1 Specifically Inhibits IL-3 Receptor Dimerization and Activation

In order to define the mechanism of BION-1 antagonism we examined BION-1for its ability to influence receptor dimerization and activation. Wehave previously shown that IL-3 or GM-CSF or IL-5 induce dimerization ofthe respective a chains with β_(c), a phenomenon that leads to receptoractivation as measured by tyrosine phosphorylation of β_(c). This isconfirmed here, with FIG. 12 showing that in the absence of cytokinesantibodies to the α chain (left panel), or β_(c) (right panel),immunoprecipitate their appropriate antigens (α chain and β_(c)respectively). In the presence of IL-3, dimerization of α and β_(c)takes place allowing either anti-α chain or anti β_(c) MoAb toimmunoprecipitate both receptor subunits. This is accompanied bytyrosine phosphorylation of β_(c) (top panel). We show in this figurethat pre-incubation of the cells with BION-1 blocks receptordimerizatidn and tyrosine phosphorylation of β_(c). As a control we usedthe anti β_(c) MoAb 1C1 which was unable to prevent receptordimerization and activation.

BION-1 Specifically Inhibits Chronic Myelomonocytic Cell Growth

BION-1 is shown to inhibit the activity of one or all of IL-5, IL-3 &GM-CSF mediated effectors of leukaemic cells. In particualr BION-1inhibits growth in vitro of chronic myelomonocytic cells (CMML), whereasa control antibody (1C1) does not (FIG. 14). Furthermore, BION-1 inhbitseven in the presence of IL-3 whereas the control does not.

Screening and Isolation of New Inhibitory Compounds

A large range of potential therapeutic compounds that might act asantagonists, or perhaps agonists of IL-3, GM-CSF and IL-5 individuallyor collectively, can be readily screened. The screening is initially todetermine whether the binding of BION-1 or a fragment thereof to β_(c)receptor or fragment is inhibited. The nature of these inhibitorycompounds will not be limited, and the methods used for a binding assaycan be any one of the many techniques known to those skilled in the art.Such methods may include affinity selection chromatography,ultrafiltration assays, the scintillation proximity assay, interfacialoptical techniques, the quartz crystal microbalance, the jet ring cell,interferometric assays using porous silicon to immobilise the receptor.Reference to such techniques can be found in Woodbury et al 1999, whichreference is incorporated herein in its entirety.

The range of therapeutic compounds may include peptides,oligonucleotides, or other small organic or inorganic molecules. FIG. 13shows the results of screening 7-mer and 12-mer peptide libraries usingsoluble β_(c) domain 4 supported on ELISA plates.

Deposit of Cell Line

The cell line BION-1 was deposited on the Apr. 29th, 1998 in theAmerican Type Culture Collection (ATCC) at 101801 University Boulevard,Manassas, Va., United States of America and has been designated ATCCHB-12525.

TABLE I Epitope mapping of Bion-1. Binding affinities of MoAb Bion-1tested on COS cells transfected with wild type βc or mutants of βcBion-1 KD 1C1 KD βc wild type: 49.3* 4.4 βc mutated in the B′-C′ loop:M363A/R364A 0† 3.8 Y365A 69.4 1.5 E366A 0 2.4 H367A 27.0 2.8 1368A 21.72.4 D369A/H370A 32.4 3.6 βc mutated in the F′-G′ loop: R418A 0 1.8 T419A23.3 3.2 G420A 53.0 1.5 Y421A 38.9 2.3 *K_(D) in nM 0† = not detectablebinding

TABLE II Inhibition of IL-5, GM-CSF and IL-3 mediated eosinophil colonyformation by BION-1 [MoAb BION-1] [MoAb 8E4] (μM) Medium (100 μM) 0.1 110 100 IL-5 (1 nM) 13 ± 4* 15 ± 3  13 ± 4  8 ± 2 2 ± 2 2 ± 0 GM-CSF 9 ±4 18 ± 4  20 ± 4  13 ± 4  2 ± 2 0 ± 0 (2 nM) IL-3 (2 nM) 4 ± 2 8 ± 1 8 ±2 4 ± 1 1 ± 1 0 ± 1 NONE 0 ± 0 0 ± 0 0 ± 0 0 ± 0 2 ± 0 0 ± 0 *Number ofday 14 eosinophil colonies per 10⁵ seeded bone marrow cells. Valuesshown are the mean from triplicate determination ± SEM.

REFERENCES

-   Allen et al 1997, Am J Otolaryngol 18:239-246-   Bagley et al 1997a, Blood 89:1471-   Bagley et al, 1997b; J Allergy Clin immunol; 99;725-728-   Barry, S. C., et al (1994) J. Biol. Chem., 269, 8488-8492.-   Bates et al, 1996; J Imunol, 156:711-718-   Contreras, M. A. et al (1983) Methods Enzymol., 92, 277-292.-   D'Andrea R J et al, (1996) Blood 87:2641-2648-   Davis, S. et al (1993) Science, 260, 1805-1810.-   Fukuda et al 1994, J Allergy Clin Immunol 94, 584-   Gearing, D. P. et al (1994) Proc. Natl. Acad. Sci. USA, 91,    1119-1123.-   Giri, J. G., et al (1994) EMBO J., 13, 2822-2830.-   Hercus, Blood (1994a) Blood, 83:3500-   Hercus, T. R. et al (1994b) Proc. Natl. Acad. Sci. USA, 91,    5838-5842.-   Hibi, M. et al (1990) Cell, 63, 1149-1157.-   Hilton, D. J. et al (1994) EMBO J., 13, 4765-4775.-   Hilton, D. J. et al (1996) Proc. Natl. Acad. Sci. USA, 93, 497-501.-   Jenkins et al (1995) EMBO J. 14:4276-   Kimura, Y. et al (1995) Int. Immunol., 7, 115-120.-   Korpelainen et al (1993) Proc Nat. Acad. Sci USA, 90, 11137-11141-   Korpelainen et al (1995) Blood 86, 176-182.-   Liu, J. et al (1992) J. Biol. Chem., 267, 16763-16766.-   Lopez et al, (1990) Int J Aller gClin Immunol 85, 99-102-   Lopez A F et al (1989). Proc Natl Acad Sci USA 86, 7022-7026.-   Mauser et al, (1995), Am J Respir Crit Care Med 152; 467-   McKinnon et al (1997), J Exp Med 186:121-129-   McConahey et al (1980) Methods Enzymol 70:210.-   Metcalf; (1986) Blood 67:257-   Munson, P. J. and Rodbard, D. (1980) Anal. Biochem., 107, 220-239.-   Nicoletti, I. et al (1991) J. Immunol. Methods, 139:271.-   Noguchi, M. et al (1993) Science, 262, 1877-1880.-   Pennica, D. et al (1995) J. Biol. Chem., 270, 10915-10922.-   Russell, S. M. et al (1993) Science, 262, 1880-1883.-   Scatchard (1949) Ann N.Y Acad Sci 51, 660-663-   Sun et al (1996) Blood 87, 83-92-   Sur et al, 1996, J Allergy Clin Immunol 97;1272-   Taga, T. et al (1992) Proc. Natl. Acad. Sci. USA, 89, 10998-11001.-   Takeshita, T. et al (1992) Science, 257, 379-382.-   Tavernier et al 1995, Proc Natl Acad Sci USA 23:5194-5198-   Vadas et al (1979) J Immunol 122, 1228-1236-   Walsh and Crumpton, (1977) Nature 269:307.-   Woodbury, C. P. et al (1999) J Chromatogr B Biomed Sci Appl    725(1):113-37.-   Woodcock et al (1994) EMBO J 13:5176-   Woodcock et al, (1996) J Biol Chem 271, 25999-26006-   Zurawski, S. M. et al (1993) EMBO J., 12, 2663-2670.

1. A method of inhibiting IL-5, IL-3 or GM-CSF mediated proliferation ofa chronic myelomonocytic cell by contacting said cell with a monoclonalantibody BION-1 or a fragment thereof capable of inhibiting binding ofcytokines IL-3, GM-CSF or IL-5 to the common receptor βc, wherein themonoclonal antibody or fragment thereof binds to both the B′-C′ loop andthe F′-G′ of domain 4 of the βc subunit, wherein said contacting resultsin inhibition of IL-5, IL-3 or GM-CSF mediated proliferation of saidcell.
 2. A method of inhibiting IL-5, IL-3 or GM-CSF mediated eosinophilactivation, eosinophil production or eosinophil survival, by contactingan eosinophil with a monoclonal antibody BION-1 or a fragment thereofcapable of inhibiting the binding of cytokines IL-3, GM-CSF or IL-5 tothe common receptor βc, wherein the monoclonal antibody or fragmentthereof binds to both the B′-C′ loop and the F′-G′ of domain 4 of the βcsubunit, wherein said contacting results in inhibition of IL-5, IL-3 orGM-CSF mediated activation, production or survival of said eosinophil.